Phosphatase inhibitor cocktail

For the quantitation of human Aβ_1-42, a sandwich antibody ELISA was performed according to the manufacturer’s instructions (KHB3441, Invitrogen). The right side of the neocortex and hippocampus were homogenized in RIPA buffer (Sigma) containing a protease (Roche diagnostics) and phosphatase inhibitor cocktail (AG Scientific). The samples were centrifuged for 15 minutes at 13000 RPM, rotated for a further 30 minutes, and centrifuged again at 4 °C for 15 minutes at 13000 RPM. The resulting supernatant was removed and stored at −80 °C for protein analysis. The protein concentrations of samples were determined using the Bradford assay. Duplicate neocortex and hippocampus samples from APP/PS1 mice (n = 5/group) were diluted in the buffer provided (1:50). Soluble human Aβ_1-42 levels were normalized to total protein levels and expressed as picogram of Aβ_1-42 content per milligram of total protein (pg/mg). Optical densities were read at 450 nm on a microplate reader (SpectraMax, Molecular Devices), and concentrations of Aβ_1-42 were determined by comparison to the standard curve using a 4-parameter algorithm.

HepG2 hepatocellular carcinoma cells were cultured in DMEM media containing 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin until the mid-logarithmic phase, then transferred to a 96-well plate (5 × 103/well). After incubation at 37 °C for 24 h, the cells were starved in DEME supplemented with 0.4% FBS for 24 h prior to treatment of the purified scvhFGF19. HepG2 cells were treated with 5 μg/mL scvhFGF19 at different concentrations for 15 min (or indicated time), followed by cell disruption with lysis buffer (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol) containing protease inhibitor cocktail (Quartett GmbH, Germany) and phosphatase inhibitor cocktail (AG Scientific, San Diego, CA, USA). Following centrifugation at 12,000× g and 4 °C for 20 min, total protein concentration of the supernatant was measured using the BCA kit (ThermoFisher Scientific, Waltham, MA, USA) and separated by SDS-PAGE. From the lysates, biological activity of scvhFGF19 was analyzed by immunoblotting with antibodies against Erk1/2 (Cell Signaling Technology, Danvers, MA, USA), phosphorylated Erk1/2 (Cell Signaling Technology), and α-tubulin (Sigma-Aldrich, St. Louis, MO, USA).